Our main objective is to study the maintenance of differentiated function in a new parathyroid in vitro gland model - the 3-dimensional organoid. Progress will apply to the second long term objective: to use the system as a model to study the development of secondary hyperparathyroidism and other diseases involving the parathyroid which results from chronic stresses to the gland. The first goal is to establish culture conditions which will maintain organoids for 2 weeks or longer in a physiological state equivalent to that of a normal parathyroid gland. In order to establish that the behavior of organoids in culture is similar to that of glands in vivo, biochemical and morphological parameters will be followed. When a control condition has been established, we will study the effects of calcium and vitamin D metabolites on the size and morphology of the organoid cells, the biosynthetic rate of parathormone, the maximum and minimum hormone secretion rates and the calcium concentration halfway between them (the set point), and the degree to which parathormone is degraded prior to secretion. Morphological and biochemical alterations due to perterbation will be examined to determine whether they are reversible or not in order to identify possible markers for a true hyperparathyroid state as opposed to the normal range of potential gland activities. Parathyroid organoids will be formed by placing parathyroid cell suspensions into culture in rotating roller tubes. Calcium and vitamin D metabolite concentrations will be changed in culture media, and the organoids will then be examined. To study biochemical parameters, organoids and culture media will be periodically examined for parathormone content, total protein, DNA, RNA, thymidine uptake, etc., and organoids additionally will be subjected to short term studies using perifusion or standard incubation procedures to estimate calcium responses, set point, and the degree of hormone degradation prior to secretion.